Cytokines in Pediatric Pilocytic Astrocytomas: a Clinico-Pathological Study

Article Cytokines in Pediatric Pilocytic Astrocytomas: A Clinico-Pathological Study

Nurfarhanah Bte Syed Sulaiman 1,2, Chik Hong Kuick 2,3, Kenneth T. E. Chang 2,3, Kai Rui Wan 1, Wen Shen Looi 4, David C. Y. Low 1,5,6, Wan Tew Seow 1,5,6 and Sharon Y. Y. Low 1,2,5,6,*

1 Department of Neurosurgery, National Neuroscience Institute, 11 Jalan Tan Tock Seng, Singapore 308433, Singapore; [email protected] (N.B.S.S.); [email protected] (K.R.W.); [email protected] (D.C.Y.L.); [email protected] (W.T.S.) 2 VIVA-KKH Paediatric Brain and Solid Tumours Laboratory, KK Women’s and Children’s Hospital, 100 Bukit Timah Road, Singapore 229899, Singapore; [email protected] (C.H.K.); [email protected] (K.T.E.C.) 3 Department of Pathology and Laboratory Medicine, KK Women’s and Children’s Hospital, 100 Bukit Timah Road, Singapore 229899, Singapore 4 Department of Radiation Oncology, National Cancer Centre, 11 Hospital Drive, Singapore 169610, Singapore; [email protected] 5 Neurosurgical Service, KK Women’s and Children’s Hospital, 100 Bukit Timah Road, Singapore 229899, Singapore 6 SingHealth Duke-NUS Neuroscience Academic Clinical Program, 11 Jalan Tan Tock Seng, Singapore 308433, Singapore * Correspondence: [email protected]

Abstract: Pilocytic astrocytomas (PCA) are WHO Grade I tumors with a favorable prognosis. Surgical resection is usually curative. Nonetheless, progressive and/or metastatic disease occurs in 20% of patients. For these patients, treatment options are limited. The role of the immune system in Citation: Bte Syed Sulaiman, N.; PCA has not previously been reported. We hypothesize that the circulating cytokines contribute to Kuick, C.H.; Chang, K.T.E.; Wan, K.R.; tumorigenicity in PCA. This is an exploratory study with a focus on the identification of circulating Looi, W.S.; Low, D.C.Y.; Seow, W.T.; cerebrospinal (CSF) cytokines associated with PCA. The primary objective is to demonstrate that Low, S.Y.Y. Cytokines in Pediatric CSF cytokines will be differentially expressed in the subset of PCAs that are difficult to treat in Pilocytic Astrocytomas: A comparison to their surgically amendable counterparts. This is a single-institution, retrospective Clinico-Pathological Study. NeuroSci study of prospectively collected data. Patients with a confirmed histological diagnosis of PCA who 2021, 2, 95–108. https://doi.org/ have simultaneous intraoperative CSF sampling are included. Cerebrospinal fluid samples are 10.3390/neurosci2010006 subjected to multiplex cytokine profiling. Patient-derived PCA lines from selected patients in the

Received: 23 December 2020 same study cohort are cultured. Their cell culture supernatants are collected and interrogated using Accepted: 5 March 2021 the sample multiplex platform as the CSF. A total of 8 patients are recruited. There were two patients Published: 10 March 2021 with surgically difficult tumors associated with leptomeningeal involvement. Multiplex profiling of the cohort’s CSF samples showed elevated expressions of IFN-γ, IL-2, IL-12p70, IL-1β, IL-4, and Publisher’s Note: MDPI stays neutral TNF-α in these two patients in comparison to the remaining cohort. Next, primary cell lines derived with regard to jurisdictional claims in from the same PCA patients demonstrated a similar trend of differential cytokine expression in their published maps and institutional affil- cell culture supernatant in vitro. Although our findings are preliminary at this stage, this is the first iations. study in pediatric PCAs that show cytokine expression differences between the two groups of PCA with different clinical behaviors.

Keywords: cerebrospinal ﬂuid; cytokines; pilocytic astrocytoma Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and 1. Introduction conditions of the Creative Commons Brain tumors are the most common solid tumors in children, comprising 25% of all Attribution (CC BY) license (https:// childhood cancers [1–3]. In this group, pilocytic astrocytomas (PCA) are the second most creativecommons.org/licenses/by/ 4.0/). common, accounting for 21 to 23% of all pediatric brain tumors [3–7]. According to the

NeuroSci 2021, 2, 95–108. https://doi.org/10.3390/neurosci2010006 https://www.mdpi.com/journal/neurosci NeuroSci 2021, 2 96

World Health Organization (WHO), PCA is classified as a Grade I tumor [3,8]. They can arise anywhere along the neuroaxis. Specifically, for PCA, the most common location is in the cerebellum, followed by the supratentorial compartment. Other places of origin include the brainstem and the spinal cord [1–3,5,9–12]. The male/female ratio is estimated to be 1.12 [11]. Broadly speaking, PCA has an extremely favorable overall prognosis with its 10-year overall survival rate as high as 96% [4,7,13]. The treatment of choice, whenever possible, is gross total surgical resection (GTR). For the majority of cases, no adjuvant radiation or chemotherapy is required [4,7]. These tumors seldom progress to higher grade gliomas, and dissemination is infrequent [14,15]. Nonetheless, recurrence and progressive disease occur in 20% of patients [7], and occasionally, tumors spread into the cerebrospinal fluid (CSF) may be encountered [7,16,17]. In this latter subset of patients, their tumors tend to be located at unresectable sites such as the optic tract, hypothalamus, and deep, midline regions adherent to important vascula- ture [7,18]. These lesions are notoriously difficult to treat, as they demonstrate a sustained tendency for progression [14] and often become a chronic disease with substantial morbidities [19]. Affected patients often suffer from life-long endocrinopathies, progressive cognitive and adaptive functioning impairments [20]. Recent studies have implicated inflammation as an important contributor in tumor behavior [21–23]. In particular, cytokines are known to coordinate various pro-inflammatory responses within the tumor microenvironment, acting on autocrine and/or paracrine pathways on both malignant and non-malignant cells [24]. Depending on the tumor microenvironment, cytokines can modulate anti-tumoral responses. Paradoxically, the same factors may induce cell transformation and malignancy instead during chronic inflammation [25]. Previous studies have demonstrated that expression changes in various cytokines have been observed in the CSF of brain tumor patients [26,27]. Nonetheless, it remains uncertain at this stage what the exact roles of these circulating CSF cytokines are in brain tumors; in particular, whether they are pro- or anti-oncogenic. Currently, we are aware of the benefits of immunotherapy for various malignancies [28,29]. Here, treatment leverages the high specificity of the immune system to target and eliminate neoplastic cells while leaving healthy cells undamaged [30]. Ex- trapolating this concept into CNS malignancies, a thorough understanding of the immune environment of brain tumors will be a requisite to identify effective immune-based treat- ments [30]. For the purposes of this study, the authors postulate that immunotherapy may be helpful for the subset of therapeutically difficult PCAs. On a separate note, brain tumors by virtue of their location, are anatomically in contact with the circulating CSF [31,32]. In addition, CSF is a readily accessible body fluid rich in immune-related factors, allow- ing it to be reflective of the pathological state of the CNS [27]. Therefore, examining the CSF of brain tumor patients is likely to yield potential cytokines of interest that provide insight into their individual tumor environments. However, there is no previous study that specifically compared CSF cytokine profiles between different PCAs. Building on this knowledge, the authors hypothesize the following: firstly, CSF cytokines are differentially expressed in the subset of PCAs that are surgically difficult to treat, in comparison to their surgically amendable counterparts; and next, PCA tumor cells will secrete these same similar cytokines into their own microenvironment.

2. Materials and Methods 2.1. Study Design and Patient Demographics This was a single-institution, retrospective study of prospectively collected data approved by the hospital ethics review board (Singhealth CIRB Ref: 2014/2079). All patients and their legal guardians provided signed informed consent for the research use of their medical data and biomaterials. This study was meant to be exploratory; with a key focus on the identiﬁcation of circulating CSF cytokines associated with PCA in the study cohort. Inclusion criteria consisted of patients who underwent surgery by the Neurosurgical Service, KK Women’s and Children’s Hospital, and had proven histological diagnosis NeuroSci 2021, 2 97

of PCA. Patients who did not have a diagnosis of PCA; those who were operated on in other hospitals, with incomplete medical records, and/or who had insufﬁcient CSF/tumor tissue were excluded. For the purposes of this study, we deﬁned 0% of tumor remnant as the achievement of gross total resection (GTR), ≤ 10% tumor remnant as near total resection (NTR), and ≥ 30% remnant as subtotal resection (STR), based on postoperative radiological results.

2.2. Tumor Histopathological Diagnosis All available archival histological glass slides, including immunohistochemical stains from the study cohort, were retrieved and reviewed by an in-house pathologist in relation to diagnostic criteria laid out in the current (2016) World Health Organization Classiﬁcation of Tumors of the Central Nervous System [9].

2.3. Cerebrospinal Fluid Collection Cerebrospinal spinal fluid was collected at the time of surgery as part of the operative procedure. For cytological examination, samples of CSF were mixed with up to 2 drops of BD Surepath™ preservative fluid (BD system, Franklin Lakes, NJ, USA), placed in a cytospin sample chamber, and centrifuged at 600 rpm for 6 min. Alcohol-fixed smears (95% ethanol for at least 60 min) were placed in an autostainer machine for Papanicolau staining, and air-dried smears were stained using the Hemacolour™ stain. Both cytospin samples and stained smears were then reviewed by our in-house pathologists for tumor cells.

2.4. Patient-Derived Tumour Cell Cultures and In Vitro Experiments Pilocytic astrocytoma tumor samples were finely minced, washed in Dulbecco’s Phos- phate Buffered Saline (Capricorn Scientific, Germany) prior to culturing in culture media containing DMEM-F12A with L-Glutamine (Capricorn Scientific, Ebsdorfergrund, Ger- many) supplemented with 10% fetal bovine serum (Capricorn Scientific, Ebsdorfergrund, Germany) and 2% actinomycin (Naclai Tesque, Kyoto, Japan). Cell cultures were main- ◦ tained in a 37 C, humidified incubator containing 5% CO2 and 95% air. Subsequently, viable cells were sub-cultured via trypsinization and expanded. Only cells cultured at less than 8 passages were used in the subsequent experiments for this study.

2.5. Molecular Investigations of Tumour and Patient-Derived Tumour Cell Lines Ribonucleic acid (RNA) was extracted from FFPE tissue using Reliaprep FFPE To- tal RNA Miniprep System (Promega, Madison WI, USA) according to the manufacturer’s instructions. Next, RNA concentration was measured by 260 nm absorbance using biophotometer plus spectrophotometer (Eppendorf, Hamburg, Germany). Approx- imately 100 to 350 ng of extracted RNA from each sample was used for the one-step RT-PCR using GoTaq 1-Step probe RT-PCR kit (Promega, Madison, WI, USA). Primers and probe flanking KIAA1549-BRAF breakpoint (68bp)–50-CGTCCACAACTCAGCCTACA-30 50-CCTCCATCACCACGAAATCCTT-30; 50-TCGGATGCCCAGACTTG-30 (probe)-were used to detect the presence of breakpoint sequence of the exon 15 of KIAA1549 and exon 9 of BRAF using Rotor-gene Q real-time cycler (Qiagen, Düsseldorf, Germany) [33]. The temperature profile for the one-step RT-PCR was 45 ◦C for 15 min, 95 ◦C for 5 min, 40 cycles of 95 ◦C for 15 s, and 60 ◦C for 1 min with yellow signal detection in every cycle at 60 ◦C. The specific amplification curve comparable to the positive control indicated the presence of the KIAA1549-BRAF (type 15-9) fusion transcript. Efforts to correlate the molecular profiles of patient-derived tumor cell lines helped to verify there was a minimal genetic drift from their original tumors [34].

2.6. Real-Time Quantitative Polymerase Chain Reaction for mRNA Expression Ribonucleic acid (RNA) was extracted from patient-derived PCA cell lines using RNeasy Mini Kit (Qiagen, Düsseldorf, Germany) according to the manufacturer’s instructions prior to lysing cells in Trizol® Reagent (Life Technologies, Carlsbad, CA, USA). NeuroSci 2021, 2 98

Reverse transcription was performed using GoScript TM Reverse Transcriptase (Promega, Madison, WI, USA) with a starting material of 1µg per sample. The prepared cDNA of 25 ng was subjected to a real-time quantitative polymerase chain reaction (RT-qPCR) using SoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories, Irvine, CA, USA) on the CFX 96TM Real-Time Detection System (Bio-Rad Laboratories, Irvine, CA, USA) according to the manufacturer’s instruction. The following primer sequence was used for the gene of interest, GFAP (forward primer: 50-CTGGAGGTTGAGAGGGACAA-30; reverse primer: 50-CAGCCTCAGGTTGGTTTCAT-30) with expression normalized against the following housekeeping gene. RPL13A (forward primer: 50- CATAGGAAGCTGGGAGCAAG- 30; reverse primer: 50-GCCCTCCAATCAGTCTTCTG-30). ACTB (forward primer: 50- TGACCCAGACATGTTTGAGA-30; reverse primer: 50- TACGGCCAGAGGCGTACGG-30) was used as an extra validation housekeeping gene. Threshold cycles (Ct) were calculated automatically using the CFX Manager™ Software (Version 3.1) (Bio-Rad Laboratories, Irvine, CA, USA).

2.7. Cytokine Multiplex Assay: CSF and Cell Supernatant Samples The LUNARISTM Human 11-Plex Cytokine Kit (AYOXXA Biosystems, Cologne, Ger- many) was used to measure cytokine concentration in the CSF and primary cell culture supernatants. This was a multiplex assay based on beads-on-a-chip technology with a classical sandwich immunoassay principle, followed by a fluorescence readout. The calibration curve was generated according to the manufacturer’s instructions. Briefly, 7 standards were prepared by serial dilution (1:4) of the Human Cytokine Standard using Assay Diluent 2. The standard consisted of the following analytes: GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 (p70), and TNF-α. The LUNARISTM BioChips were first washed. Following that, 5 µL of standard, blank, and experimental samples, which were pre-diluted in Assay Diluent 2 (1:2), were loaded into the appropriate wells. After incubation of approximately 3 h at room temperature, the BioChips were washed again. Next, 10 µL of detection antibody solution was added per well and incubated for 60 min at room temperature. The BioChips were washed again, followed by the addition of 10 µL of streptavidin-phycoerythrin reagent per well. After 30 min of incubation, the final washing step was performed. The BioChips were then air-dried and imaged using the LUNARISTM Reader (LRS-001). The quantification of the readout was performed using the LUNARISTM Analysis Suite Software. Protein concentration was reported as ρg/mL for each analyte.

2.8. Statistical Analysis All CSF- and cell supernatant experiments were performed in a minimum of 3 technical replicates. Data were presented as mean ± standard deviation (SD). Statistical analyses were performed using either Analysis of Variance (ANOVA) test or a Student’s t-test, depending on the analysis required. Differences between sample means were considered statistically signiﬁcant when the p-value was less than 0.05 (*) or less than 0.001 (**). Graphs were generated using GraphPad Prism version 8.3.1 (GraphPad Software, SD, USA).

3. Results 3.1. Study Recruitment Based on the study’s criteria, a total of 8 patients were recruited. There were 5 males and 3 females. Their ages ranged from 1 year to 19 years old (median age 9.88 years old). Six patients underwent either GTR or NTR for their tumors. There were 2 patients with leptomeningeal disease (LMD) at the time of diagnosis (Patients 2 and 5). In comparison to the others, these two patients (Patients 2 and 5) were challenging to manage, and their clinico-pathological details were described in the following as illustrative cases (Table1). NeuroSci 2021, 2, FOR PEER REVIEW 5

to the others, these two patients (Patients 2 and 5) were challenging to manage, and their clinico-pathological details were described in the following as illustrative cases. (Table 1). NeuroSci 2021, 2 99 Table 1. Summary of study cohort’s clinico-pathological information. Of notes, Patient 2 is labeled as 2a^ and 2b^, respectively, as she underwent two surgeries. Patient 5 (labeled as 5*) was first diagnosed 10 years earlier. (Abbreviation: EOR = extent of resection). Table 1. Summary of study cohort’s clinico-pathological information. Of notes, Patient 2 is labeled as 2aˆ and 2bˆ, respectively, as she underwent two surgeries. Patient 5 (labeled as 5*) was ﬁrst diagnosed 10 years earlier.Adjuvant (Abbreviation: EOR = extent Presence of KIAA- BRAF V600E LMD CSF Cytology Patientof resection). Treatment before Location of Tumor BRAF Fusion Gene Mutant Posi- (Yes/ Positive for Tumour Number Presence of SurgeryAdjuvant CSF Cytology (Yes/No) BRAFtive V600E (Yes/No) No) Cells (Yes/No) Location of KIAA-BRAF LMD Treatment before Positive for Patient Number Mutant Positive (Yes/No) Tumor Fusion Gene (Yes/No) Surgery Tumour Cells (Yes/No) 1 Suprasellar (Yes/No)No No No No (Yes/No) No(Yes/No) 2a^ Left frontotemporal Yes (15-9) Yes Yes No No 1 Suprasellar No No No No No 2b^ Left frontotemporalLeft Yes (15-9) Yes Yes Yes (Chemotherapy) No 2aˆ Yes (15-9) Yes Yes No No 3 Thirdfrontotemporal ventricle No Yes No No No Left Yes 2bˆ Yes (15-9) Yes Yes No 4 Thirdfrontotemporal ventricle No No No No(Chemotherapy) No 3Right thalamus, Third ventricle brain- No Yes No No No 4 Third ventricle No No NoYes No No 5* stem, andRight temporopari- thalamus, No Yes Yes No (Radiation) Yes 5* brainstem, and No Yes Yes No etal (Radiation) 6 Posteriortemporoparietal fossa No No No No No 6 Posterior fossa No No No No No 7 7 Posterior Posterior fossa fossa Yes Yes (15-9) (15-9) No No No No No NoNo No 8 8 Posterior Posterior fossa fossa Yes Yes (16-9) (16-9) No No No No No NoNo No

3.1.1.3.1.1. Patient Patient 2: 2: Large Large Frontotemporal Frontotemporal PCA PCA with with Intratumoral Intratumoral Arterio-Venous Arterio-Venous Shunting AA 3-year-old 3-year-old female female presented presented with with cachexia, cachexia, progressive progressive loss loss of of vision, vision, and and devel- devel- opmentalopmental delay. delay. Magnetic Magnetic resonance resonance imaging imaging (MRI) (MRI) of of her her neuroaxis neuroaxis reported reported a a large, large, heterogeneouslyheterogeneously enhancing enhancing mass mass centered centered in in the the left left frontotemporal frontotemporal lobe lobe extending extending into into thethe right right frontal frontal lobe lobe and and left left orbit, orbit, with with central central necrosis necrosis and and intratumoral intratumoral arterio-venous arterio-venous shunting.shunting. There There was was also also a a nodular nodular leptomenin leptomeningealgeal extension extension into into the the internal internal auditory auditory canal.canal. Significant Signiﬁcant mass mass effect effect with with midline midline sh shiftift to to the the right right and and obstructive obstructive hydrocephalus hydrocephalus waswas seen. seen. She underwent twotwo tumortumor debulkingdebulking surgeries surgeries approximately approximately 6 months6 months apart. apart. In Inbetween between the the surgeries, surgeries, she she did did chemotherapy, chemotherapy, consisting consisting of carboplatin of carboplatin and and vincristine vincristine [32]. [32].The secondThe second surgery surgery proceeded proceeded with anotherwith another 40% of 40% the of tumor the tumor being removed.being removed. Signiﬁcant Sig- nificantblood loss blood and loss hemodynamic and hemodynamic instability instability halted the halted surgery. the Thesurgery. decision The wasdecision made was not madeto proceed not to inproceed view of in the view patient’s of the safety.patient’s She safety. has sinceShe has recovered since recovered back to herback baseline to her baselineneurological neurological status (Figure status1 (Figure). 1).

Figure 1. Representative T1-weighted, post-contrast MRI images of Patient 2’s tumor in axial (A) and sagittal (B) views, Figurerespectively. 1. Representative They depict T1-weighted, a large, frontotemporal post-contrast heterogeneously MRI images of enhancing Patient 2′s solid-cystic tumor in axial tumor (A) extending and sagittal into (B the) views, right respectively.frontal lobe and They left depict orbit. a Significant large, frontotemporal mass effect withheterogene right midlineously enhancing shift and obstructivesolid-cystic hydrocephalustumor extending is alsointo observed.the right frontal(C) A hemotoxylin lobe and left andorbit. eosin-stained Significant mass photomicrograph effect with righ oft Patientmidline 2’s shift from and her obstructive first surgery. hydro Thiscephalus is a PCA is thatalso comprisesobserved. bipolar cells with bland nuclei. Rosenthal fibers are present. (magnification ×400). NeuroSci 2021, 2, FOR PEER REVIEW 6

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(C) A hemotoxylin and eosin-stained photomicrograph of Patient 2′s from her first surgery. This is a PCA that comprises bipolar cells with bland nuclei. Rosenthal fibers are present. (magnification × 400). 3.1.2. Patient 5: Progressive Intraventricular PCA with Leptomeningeal Disease 3.1.2. ThisPatient is a5: previously Progressive well Intraventricular 9-year-old male PCA who with initially Leptomeningeal presented withDisease an unsteady gait,This left is hemiparesis, a previously and well symptoms 9-year-old of male raised who intracranial initially presented pressure. with An an urgent unsteady MRI gait,demonstrated left hemiparesis, a multi-lobulated and symptoms cystic of lesion raised involving intracranial the pressure. right thalamus, An urgent midbrain, MRI demonstratedand hemipons. a multi-lobulated There was also cystic tumor lesion extension involving across the the right right thalamus, cerebellar midbrain, peduncle and to hemipons.the right temporoparietal There was also tumor lobe. extension Areas of across nodular the enhancementright cerebellar were peduncle seen. to Resultant the right temporoparietalhydrocephalus associated lobe. Areas with of nodular periventricular enhancement lucency were was seen. observed. Resultant Subtotal hydrocephalus excision of associatedthe lesion andwith insertion periventricular of a ventriculoperitoneal lucency was observed. shunt Subtotal was performed. excision of Histopathology the lesion and insertionreported of a pilocytica ventriculoperitoneal astrocytoma with shunt visible was performed. inﬁltration Histopathology into pia-arachnoid reported layers. a Ki-67pilo- cyticindex astrocytoma was 1 to 2% with (Figure visible2). Theinfiltration tumor remnantinto pia-arachnoid was subjected layers. to intensity-modulatedKi-67 index was 1 to radiation therapy (IMRT) of 42 Gy over fractionated into 25 sessions. Approximately 2% (Figure 2). The tumor remnant was subjected to intensity-modulated radiation therapy 10 years later, surveillance scans showed a solid-cystic lesion in the right postero-temporal (IMRT) of 42 Gy over fractionated into 25 sessions. Approximately 10 years later, surveil- region, associated with extensive leptomeningeal disease involving the skull base, along lance scans showed a solid-cystic lesion in the right postero-temporal region, associated the optic apparatus, supra- and infratentorially (Figure3). The decision was made for with extensive leptomeningeal disease involving the skull base, along the optic apparatus, excision of the right cerebellopontine lesion for histological reassessment and adjuvant supra- and infratentorially. (Figure 3). The decision was made for excision of the right chemotherapy after. cerebellopontine lesion for histological reassessment and adjuvant chemotherapy after.

Figure 2. 2. RepresentativeRepresentative MRI MRI brain brain images images of of Patient Patient 5 5 in in ( (AA)) T2-weighted, T2-weighted, axial, axial, and and ( (BB)) T1-weighted, T1-weighted, post-contrast post-contrast views views when he was first diagnosed at 9 years old. There is a lobulated cystic mass with minimal solid components involving the when he was first diagnosed at 9 years old. There is a lobulated cystic mass with minimal solid components involving right thalamus, midbrain, hemipons, cerebellar peduncle, and medial temporal lobe. Obstructive hydrocephalus is also the right thalamus, midbrain, hemipons, cerebellar peduncle, and medial temporal lobe. Obstructive hydrocephalus is noted. (C) A hemotoxylin and eosin-stained photomicrograph of Patient 5′s tumor during his first surgery. The lesion comprisesalso noted. bipolar (C) A hemotoxylincells with bland and nu eosin-stainedclei. Eosinophilic photomicrograph granular bodies of Patient are present. 5’s tumor (magnification during his first × 400). surgery. The lesion comprises bipolar cells with bland nuclei. Eosinophilic granular bodies are present. (magnification ×400). NeuroSci 2021, 2 101 NeuroSci 2021, 2, FOR PEER REVIEW 7

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FigureFigure 3. 3.Representative Representative MRI MRI brain brain imagesimages ofof PatientPatient 5 pe performedrformed 10 10 years years after after his his initial initial diagnosis. diagnosis. (A) ( AT1-weighted,) T1-weighted, post-contrast image in axial view shows multiple confluent intra-axial multiloculated cystic lesions with enhancing solid post-contrast image in axial view shows multiple confluent intra-axial multiloculated cystic lesions with enhancing solid Figurecomponents 3. Representative involving the MRI right brain thalamus, images basa of Patientl ganglia, 5 pefrontal,rformed temporal, 10 years and after midbrain. his initial (B )diagnosis. T1-weighted, (A) T1-weighted,post-contrast componentspost-contrastimage in axial involving image view inwith the axial righta solid-cystic view thalamus, shows le multiplesion basal along ganglia, confluent the right frontal, intra-axial cerebellopontine temporal, multilocula and lesi midbrain.tedon (yellowcystic (lesionsB arrow).) T1-weighted, with This enhancing lesion post-contrast was solid ex- imagecomponentscised in in axial entirety viewinvolving for with reduction the a solid-cystic right of thalamus,local lesion mass basa effect alongl ganglia, and the righthistological frontal, cerebellopontine temporal, reassessment. and lesion midbrain. (yellow (B arrow).) T1-weighted, This lesion post-contrast was excised image in axial view with a solid-cystic lesion along the right cerebellopontine lesion (yellow arrow). This lesion was ex- in entirety for reduction of local mass effect and histological reassessment. cised in entirety for reduction of local mass effect and histological reassessment. 3.2. Selected CSF Cytokines Are Differentially Expressed in Therapeutically Challenging PCAs 3.2. Selected CSF Cytokines Are Differentially Expressed in Therapeutically Challenging PCAs 3.2. SelectedMultiplex CSF profiling Cytokines of Are CSF Differentially specimens Expressedfrom the in8 patientsTherapeutically observed Challenging statistically PCAs sig- nificantlyMultiplex higher profiling expressions of CSF of specimensIFN-γ, IL-2, from IL-12p70, the 8 IL-1 patientsβ, IL-4, observed and TNF- statisticallyα for Patients signifi- Multiplex profiling of CSF specimens from the 8 patients observed statistically sig- cantly2 and 5, higher in comparison expressions to the of other IFN-γ patients., IL-2, IL-12p70, The remaining IL-1β ,cytokines IL-4, and GM-CSF, TNF-α for IL-5, Patients IL-6, 2 nificantly higher expressions of IFN-γ, IL-2, IL-12p70, IL-1β, IL-4, and TNF-α for Patients andand 5,IL-8 in comparisondemonstrated to equivocal the other findings; patients. neit Theher remaining did they show cytokines any trend GM-CSF, nor statistical IL-5, IL-6, 2 and 5, in comparison to the other patients. The remaining cytokines GM-CSF, IL-5, IL-6, andsignificance. IL-8 demonstrated (Figure 4). equivocalOf note, results findings; from neither a separate did they study show with any CSF trend collected nor statistical from and IL-8 demonstrated equivocal findings; neither did they show any trend nor statistical significancenon-tumor patients (Figure were4). Of used note, to corroborate results from these a separate findings. study (Appendix with CSFA and collected Figure A1). from significance. (Figure 4). Of note, results from a separate study with CSF collected from non-tumor patients were used to corroborate these findings (AppendixA and Figure A1). non-tumor patients were used to corroborate these findings. (Appendix A and Figure A1).

Figure 4. Graph results of CSF cytokine profiling that show differential expression with statistical significance for Patients 2 and 5 versus the rest of the patients. These include (A) IFN-γ, (B) IL-2, (C) TNF-α, (D) IL-12p70, (E) IL-1β, and (F) IL-4. Figure 4. Graph results of CSF cytokine profiling that show differential expression with statistical significance for Patients Figure(Abbreviation: 4. Graphresults N.D. = ofnot CSF detected) cytokine.* p proﬁling< 0.05; ** p that < 0.001. show differential expression with statistical signiﬁcance for Patients 2 2 and 5 versus the rest of the patients. These include (A) IFN-γ, (B) IL-2, (C) TNF-α, (D) IL-12p70, (E) IL-1β, and (F) IL-4. and 5 versus the rest of the patients. These include (A) IFN-γ,(B) IL-2, (C) TNF-α,(D) IL-12p70, (E) IL-1β, and (F) IL-4. (Abbreviation: N.D. = not detected).* p < 0.05; ** p < 0.001. (Abbreviation: N.D. = not detected). * p < 0.05; ** p < 0.001. NeuroSciNeuroSci 20212021,, 22, FOR PEER REVIEW 1028

3.3.3.3. Patient-Derived Patient-Derived Tumour Tumour Cells Cells Express Express PCA PCA Molecular Molecular Markers Markers and and Secrete Secrete Cytokines Cytokines in VitroIn Vitro Patient-derivedPatient-derived PCA PCA cell cell lines lines were were successf successfullyully cultured from Patients 2 (both(both surg-surgeries),eries), 3,3, andand 5.5. TheyThey were examined for GFAP mRNA expression and KIAA1549-BRAF genegene fusion. fusion. The The results results concurred concurred with with the thepatients’ patients’ corresponding corresponding primary primary tumors tumors (Ta- ble(Table 2). Following2) . Following that, that,cytokine cytokine profiling proﬁling of their of cell their supernatants cell supernatants in culture in culture demonstrated demon- similarstrated trends similar to trends the corresponding to the corresponding tumors, tumors, except except for IL-4. for However, IL-4. However, only IL-12p70 only IL-12p70 sta- tisticallystatistically significant signiﬁcant for forPatient Patient 2 (cell 2 (cell lines lines from from both both surgeries) surgeries) and and Patient Patient 5. 5. For For this this experiment,experiment, cytokine cytokine expression expression was was compared compared to to the the blank blank (cell (cell culture culture media media only) only) for for thethe statistical statistical analysis. analysis (Figure 5 5).).

TableTable 2. Summary of molecular markers validated validated in in primary primary cell cell lines lines in in tandem tandem with with thei theirr corresponding corresponding tumors. tumors. For For GFAPGFAP expression, immunohistochemistryimmunohistochemistry was was used used for for the the tumors tumo asrs part as part of routine of routine histological histological examination. examination. GFAP mRNAGFAP mRNAexpression expression using RT-qPCR using RT-qPCR was used was for used the cellfor the lines cell as lines described as described in ‘Materials in ‘Materials and methods’. and methods’. Primary Tumor Ex- Patient Primary Tumor PrimaryPositive Tumor Patient-DerivedPatient-Derived Cells Posi- Cells Primary Tumor Patient-DerivedPatient-Derived Cells Cells Ex- Patient Number Positive for Positive for pressesExpresses GFAP GFAP Express GFAP Number for KIAA-BRAF (Yes/No) tive for KIAA-BRAF (Yes/No) press GFAP (Yes/No) KIAA-BRAF (Yes/No) KIAA-BRAF (Yes/No) (Yes/No)(Yes/No) (Yes/No) 2a 2a Yes (15-9) Yes (15-9) Yes (15-9) Yes (15-9) Yes Yes Yes Yes 2b 2b Yes (15-9) Yes (15-9) Yes (15-9) Yes (15-9) Yes Yes Yes Yes 3 No No Yes Yes 3 No No Yes Yes 5 No No Yes Yes 5 No No Yes Yes

Figure 5. Graph results of in vitro cell culture supernatant cytokine profiling that show differential expressions for Patients Figure 5. Graph results of in vitro cell culture supernatant cytokine profiling that show differential expressions for Patients 2 and 5, versus the rest of Patient 3 and a blank (cell culture media only). These include (A) IFN-γ, (B) IL-2, (C) TNF-α, (D) γ α IL-12p70,2 and 5,versus (E) IL-1 theβ, restand of(F) Patient IL-4. After 3 and analysis, a blank only (cell IL-12p70 culture media demonstrates only). These statistical include significance (A) IFN- ,(thatB) is IL-2, similar (C) TNF-to the , CSF(D) IL-12p70,findings. * (E p) < IL-1 0.05;β, ** and p < ( F0.001.) IL-4. After analysis, only IL-12p70 demonstrates statistical significance that is similar to the CSF findings. * p < 0.05; ** p < 0.001.

NeuroSci 2021, 2 103

4. Discussion For the majority of pediatric PCA, maximal safe surgical resection is recommended as the first-line treatment for tumors in areas of the brain amenable to complete removal [7,10]. Advancements in research have provided molecularly targeted diagnosis and treatment of PCA [2]. Examples include the identification of the KIAA1549-BRAF fusion gene in the majority of PCAs [35,36] and BRAF V600E mutation in the minority [37]. Nonetheless, targeted treatment with regards to the subset of surgically challenging PCAs, as highlighted in our cohort, remains a significant clinical gap. Specifically, for children with brain tumors who survive their disease after surgery and adjuvant treatment, many have long-term cognitive disabilities that limit their ability to live independently, progress fully in their education, or pursue a vocation [38]. For many of such patients, these post-chemoradiation treatment morbidities usually manifest long after the primary tumor is cured, even beyond the fourth decade of life [39]. Reports in the literature show that childhood cancer survivors’ brains suffer progressive neurological deficits in visual processing [40,41], visual-motor functioning [42], attention and executive functioning [43]. Given that these so-called ‘low- grade’ tumors behave as a chronic disease with an indolent course, it is hence imperative that the choice of therapeutic intervention must be balanced against its potential for impact on the child’s long-term growth and development [44]. Neuroinflammation has been implicated as an important player in tumor development [21–23]. In particular, cytokines and their inflammation-related partners have been demonstrated to coordinate pro-inflammatory responses within the tumor microenvironment, acting on autocrine, and/or paracrine pathways on malignant and non-malignant cells [24]. Broadly speaking, cytokines are small glycoproteins that bind to cell surface receptors and regulate the development, survival, and function of immune cells. Evolv- ing data has observed their involvement in various intracranial pathologies, including traumatic head injury, post-hemorrhagic hydrocephalus, demyelination disease and so forth [45,46]. Although cytokines are ubiquitous, their individual expressions vary according to the context of the disease and state of each patient’s immune system. Specifically, in brain tumors, cytokines have been extensively studied as potential therapeutic agents to manipulate the immune response to tumor cells [47]. In recent years, we are now aware that they can also function in the tumor microenvironment to induce cell proliferation, invasion, angiogenesis, and suppression of certain immune functions [48]. The latter postulates that since the CNS is not an immune-privileged site, therefore, regulatory immune cells and cytokines protect against excessive inflammation that involves the brain [49]. Aspects of neuroinflammation will vary within the context of disease, injury, infection, or stress [50]. It follows that, as brain tumors progress, perilesional tissue damage and hypoxia will induce regulatory cytokines and immune cells to counteract inflammation [51,52]. Putting it altogether, these factors can firstly contribute to the immunosuppressive behavior of the tumor itself and, next, blunt an anti-tumor immune response. At this point in time, the option of immunotherapy is not available to the subset of PCA patients whose tumors are difficult to eradicate. Understanding the immune environment in which pediatric brain tumors exist is a first step to identifying effective immune-based therapies for these diseases [30]. As no matched normal brain tissue is collected as part of the surgical procedure, we did not compare cytokine expression between tumor cells and their non-tumor counterparts for each patient. More importantly, the immune system response is disease-context dependent. Under such circumstances, cytokine expressions are expected to overlap in different medical conditions. For the purposes of our study, the key aim is to assess for differences in cytokine expression between surgically amendable PCAs and those that are not. A blank media control is used to ensure there is no background expression from the growth factors in the cell culture media. Putting it together, we are able to demonstrate that firstly, patients with PCAs have circulating cytokines in their CSF; and selected cytokines have higher expression in the two patients with tumors that were difficult to treat. Following that, we demonstrated that similar cytokines are expressed by the patient-derived cell via an in vitro approach. NeuroSci 2021, 2 104

One such cytokine is interferon-gamma (IFN-γ), a pleiotropic, homodimer molecule formed by the noncovalent association of two 17 kDa polypeptide subunits [53]. This cytokine is considered to be a major effector of immunity and has been traditionally associated with anti-proliferative, pro-apoptotic, and anti-tumor mechanisms [54]. Nonetheless, there is growing evidence that IFN-γ facilitates contributing to tumor initiation by firstly promoting changes in tumor cell phenotype towards increased fitness for growth in the immunocompetent host; and next, contributing to homeostatic or cancer-triggered mechanisms to establish an immunosuppressive tumor microenvironment [55,56]. Currently, biological mechanisms by which IFN-γ functions are not fully understood, it is likely that the effect depends on multiple processes. IFN-γ primarily activates the JAK–STAT pathways that lead to the induction of the expression of multiple genes. In cancer cells, alterations in gene expression that are caused by IFN-γ are presumably associated with increased immunogenicity, which thereby induces immune stimulation [56]. Furthermore, some studies have established that IFN-γ is closely related to IL-12, which triggers NK and T cells, favoring cell-mediated immunity [57–59]. This is an interesting relationship that our current study intends to pursue in-depth to look for potential translational targets. In tandem with IFN-γ, other cytokines produced in chronic inflammation, such as TNF-α and IL1-β, are also reported to promote cancer growth and progression. For instance, as a pro-inflammatory cytokine, TNF-α is secreted by inflammatory cells, which may be involved in inflammation-associated carcinogenesis [60]. These cytokines can steer cells toward more malignant phenotypes via multiple mechanisms, including induction of DNA damage response, angiogenesis, and activation of signaling pathways that promote cancer cell survival and, or proliferation [61,62]. In contrast to the rest, IL-2 plays a vital role in the growth as well as differentiation of T cells, B cells, natural killer cells, and many other cell types [61]. IL-2 is a T cell-derived common cytokine and was the first cytokine to be successfully used in the treatment of cancer. This was because it can promote T cells as well as NK cells. IL-2 can induce T cell proliferation, differentiation, and activation [62]. Overall, more research is needed to understand the immuno-mechanisms of PCAs, which are crucial pre-requisites for treatment optimization. Although this is a retrospective study with limited numbers, it is a novel attempt at exploring the possibility of immune factors involved in the oncogenesis of PCAs. Results from this proof-of-concept study allow us to pursue in-depth studies focusing on elucidating the relationship between PCAs and their corresponding cytokines of interest. The future work for this project includes, firstly, the comprehensive interrogation of each patient’s tumor to assess for immune infiltration will be corroborated with the circulating CSF cytokines to identify possible relationships between them. Another consideration is to compare the immune profiles between the subset of challenging PCAs and other high-grade astrocytomas. Next, functional studies are performed to validate their biological significance in PCA tumorigenesis. In addition, a larger, prospective study that also includes the collection of blood from each patient as well, is already in place.

5. Conclusions In summary, our study demonstrates there is differential cytokine expression in PCAs that are difficult to treat in comparison to those that are surgically amendable. Although our findings are preliminary, this is the first dedicated study exploring the involvement of immune-related factors in pediatric PCAs. This is important as a first step for further investigation of their translational role, especially in a subset of challenging pediatric PCAs. In the meantime, the authors advocate continued efforts with international collaborators to work together for better disease understanding in affected children. NeuroSci 2021, 2 105

NeuroSci 2021, 2, FOR PEER REVIEW 11

Author Contributions: Conceptualization, N.B.S.S. and S.Y.Y.L.; methodology, N.B.S.S., C.H.K., K.T.E.C.Author andContributions: S.Y.Y.L.; software, Conceptualization, N.B.S.S., C.H.K. N.B.S.S. and K.T.E.C.;and S.Y.Y.L.; validation, methodology, N.B.S.S. N.B.S.S., and C.H.K.; C.H.K., formal analysis,K.T.E.C., N.B.S.S. and S.Y.Y.L.; and S.Y.Y.L.; software, investigation, N.B.S.S., C.H.K., N.B.S.S. and and K.T.E.C.; S.Y.Y.L.; validation, resources, N.B.S.S. D.C.Y.L., and W.T.S.,C.H.K.; K.T.E.C. for- andmal S.Y.Y.L.; analysis, data N.B.S.S. curation, and S.Y.Y.L.; K.R.W., W.S.L.,investigation, N.B.S.S. N.B.S.S. and C.H.K.; and S.Y.Y.L.; writing—original resources, D.C.Y.L., draft preparation, W.T.S., K.R.W.,K.T.E.C., N.B.S.S. and S.Y.Y.L.; and S.Y.Y.L.; data curation, writing—review K.R.W., W.S. andL., N.B.S.S., editing, and K.T.E.C. C.H.K.; and writing—original S.Y.Y.L.; visualization, draft S.Y.Y.L.;preparation, supervision, K.R.W., N.B.S.S., S.Y.Y.L.; and project S.Y.Y.L.; administration, writing—review S.Y.Y.L.; and editing, funding K.T.E.C. acquisition, and S.Y.Y.L.; D.C.Y.L. vis- and K.T.E.C.ualization, All authorsS.Y.Y.L.; havesupervision, read and S.Y.Y.L.; agreed toprojec the publishedt administration, version S.Y.Y.L.; of the manuscript. funding acquisition, D.C.Y.L. and K.T.E.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by VIVA-KKH Paediatric Brain and Solid Tumour Programme. ThisFunding: is a philanthropic This research was grant funded that wasby VIVA-KKH awarded toPaed theiatric institution Brain and (KK Solid Women’s Tumour andProgramme. Children’s Hospital)This is a philanthropic where the study grant was that conducted. was awarded to the institution (KK Women’s and Children’s Hos- pital) where the study was conducted. Institutional Review Board Statement: The study was conducted according to the guidelines of the DeclarationInstitutional of Review Helsinki, Board and Statement: approved byThe the study Institutional was conducted Review according Board (orto the Ethics guidelines Committee) of the of Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of SingHealth Centralised Institutional Review Board (protocol code Singhealth CIRB Ref: 2014/2079 SingHealth Centralised Institutional Review Board (protocol code Singhealth CIRB Ref: 2014/2079 and 20 May 2020). and 20 May 2020). InformedInformed Consent Consent Statement: Statement:Informed Informed consent consent was was obtained obtained from from all all subjects subjects involved involved in thein the study. Datastudy. Availability Statement: Not applicable. ConﬂictsData Availability of Interest: Statement:The authors Not declare applicable. no conﬂict of interest. The funders had no role in the design ofConflicts the study; of in Interest: the collection, The authors analyses, declare or interpretation no conflict of ofinte data;rest. in The the funders writing had of the no manuscript, role in the or indesign the decision of the study; to publish in the the collection, results. analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Appendix A Appendix A This is a separate, ongoing study based in the same institution. As part of this study’sThis protocol, is a separate, CSF ongoing from non-tumour study based patients in the same after institution. informed As consent part of this are study’s included. Theseprotocol, are patientsCSF from who non-tumour require CSF patients diversion after surgeriesinformed forconsent other are intracranial included. These pathologies. are Theypatients include who 3require patients CSF diagnosed diversion surgeries with post-traumatic for other intracranial hydrocephalus pathologies. (1), They congenital in- hydrocephalusclude 3 patients (1), diagnosed and post-haemorrhagic with post-traumat hydrocephalusic hydrocephalus (1). Once(1), congenital again, CSF hydroceph- samples are interrogatedalus (1), and via post-haemorrhagic a multiplex immunoassay hydrocephalus kit that (1). includesOnce again, the CSF cytokines samples of are interest interro- from thegated main via study. a multiplex Here, samples immunoassay are prepared kit that in in duplicatescludes the forcytokines each patient of interest and examined from the as permain the study. manufacturer’s Here, samples instructions. are prepared Results in du areplicates tabulated, for each analysed patient and and compared examined against as per the manufacturer’s instructions. Results are tabulated, analysed and compared against the samples of interest from the main study (namely, Patients 2a, 2b and 5) (Figure A1). the samples of interest from the main study (namely, Patients 2a, 2b and 5). (Figure A1).

Figure A1. Graph showing comparison of the CSF cytokine expression combinatorial raw values between the non-tumour Figure A1. Graph showing comparison of the CSF cytokine expression combinatorial raw values between the non-tumour samples and surgically challenging PCA samples (Patients 2a, 2b and 5). * p < 0.05. samples and surgically challenging PCA samples (Patients 2a, 2b and 5). * p < 0.05. NeuroSci 2021, 2 106

In this subgroup investigation, we observe differentially expressed CSF cytokines between non-tumour samples versus the patients with the surgically challenging PCAs. For IFN-γ, IL-12p70, IL-2 and TNF-α, their expressions in the non-tumour CSF patients are lower than those in the surgically challenging PCAs. This trend is similar to the results in Figure4 from the main study. In contrast, IL1- β and IL-4 have higher expression in the non-tumour group versus the surgically challenging group. Given that firstly, immune-mediated expression is context-dependent in neuroinflammation; and next, cytokine expressions tend to vary between different intracranial pathologies, our findings are not entirely unexpected [45,46]. Nonetheless, the observations from this subgroup analysis suggest that there are distinct cytokines in the surgically challenging PCAs, that differ from our non-tumour CSF cohort and the PCA patients amenable to treatment.

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